[show abstract][hide abstract] ABSTRACT: Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle for further understanding the regulatory roles of reversible phosphorylation in this organism. In this study, we used high accuracy mass spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and 2D-LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network reveals the potential biological functions for many previously unannotated proteins and predicts several putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila by using a phosphoproteomic approach, and suggests a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.
[show abstract][hide abstract] ABSTRACT: Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis, and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain and a proline-rich (PXXP) repeat, all of which mediate binding to different partners. To elucidate BAG3 interaction network at the molecular level, we employed QUICK (quantitative immunoprecipitation combined with knockdown) and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through down-modulation of apoptosis. Taken together, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach.
[show abstract][hide abstract] ABSTRACT: Microcystin-LR (MCLR) is a widespread cyanotoxin and has immunotoxicity to animals, including fish. Chemokines are considered to play important roles in inflammatory response induced by MCLR. In this study, we cloned the full-length cDNA of interleukin-8 (IL-8) from bighead carp (Aristichthys nobilis) for the first time. The full-length IL-8 cDNA was 552 bp and contained a 297-bp open-reading frame that encoded for a 98-amino acid protein. The deduced IL-8 protein had a typical aspartic acid (D)-leucine (L)-arginine (R) and a CXC motif at the N-terminal, which were conserved in most fish species. Phylogenetic analysis showed that bighead carp IL-8 protein was grouped in the teleost IL-8 lineage 2. Under normal conditions, the expression of IL-8 is constitutive and weak in all tested tissues. However, MCLR treatment could significantly increase the transcription of IL-8 in bighead carp in a temporal- and dose-dependent pattern. The present study will help us to understand more about the evolution of IL-8 and its function in the MCLR induced proinflammatory response in bighead carp.
Archives of Environmental Contamination and Toxicology 06/2013; · 2.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria, and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high number of protein kinases and phosphatases with specificity on Ser, Thr and Tyr residues. However, only a few dozen phosphorylation sites in cyanobacteria are known, presenting a major obstacle for further understanding the regulatory roles of reversible phosphorylation in this group of bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the model cyanobacterium Synechococcus sp. PCC 7002. In total, 280 phosphopeptides and 410 phosphorylation sites from 245 Synechococcus sp. PCC 7002 proteins were identified through the combined use of protein/peptide prefractionation, TiO2 enrichment and LC-MS/MS analysis. The identified phosphoproteins were functionally categorized into an interaction map and found to be involved in various biological processes such as two-component signaling pathway and photosynthesis. Our data provides the first global survey of phosphorylation in cyanobacteria by using a phosphoproteomic approach, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may help reveal the physiological functions underlying Ser/Thr/Tyr phosphorylation and facilitate the elucidation of the entire signaling networks in cyanobacteria.
Journal of Proteome Research 03/2013; · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small noncoding RNAs, approximately 22 nucleotides in length, found in diverse organisms. They have emerged in recent years as key regulators of a broad spectrum of cellular functions. miRNAs regulate biological processes by inducing translational inhibition and degradation of their target mRNAs through base pairing to partially or fully complementary sites. In the field of miRNA research, the identification of the targets of individual miRNAs is of utmost importance. Our understanding of the molecular mechanisms by which individual miRNAs modulate cellular functions will remain incomplete until a full set of miRNA targets is identified and validated. Since a miRNA may regulate many of its targets at the translational level without affecting mRNA abundance, proteomic methods are best suited for revealing the full spectrum of miRNA targets. Quantitative proteomics is emerging as a powerful toolbox for identifying miRNA targets and for quantifying the contribution of translational repression by miRNAs. In this review, the authors summarize the quantitative proteomic approaches that have been employed for identification of miRNA targets and discuss current challenges as well as possible ways of overcoming them.
Expert Review of Proteomics 10/2012; 9(5):549-59. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microcystin-LR (MCLR) is a widespread cyanotoxin produced in algal blooms, and has potent hepatotoxicity and tumor-promoting activity. We cloned the full-length cDNA of Id1 in bighead carp. The full-length Id1 cDNA was 954bp and contained a 387bp ORF. Bighead carp Id1 shared high identity with zebrafish Id1 amino acid sequence, and phylogenetic analysis showed that teleost Id1 evolved closely. Bighead carp Id1 constitutively expressed in all tested tissues in normal. When tested at two different time points post exposure and at 3 different MCLR doses, Id1 expression increased in a time-dependent pattern, and Id1 expression in brain was very sensitive to MCLR exposure. The present study will help us to understand more about the evolution of Id1 molecule and its role in the MCLR induced cell differentiation and cancer promoting in bighead carp.
Environmental toxicology and pharmacology. 05/2012; 34(2):324-333.
[show abstract][hide abstract] ABSTRACT: Substantial evidence indicates that microRNA-21 (miR-21) is a key oncomiR in carcinogenesis and is significantly elevated in multiple myeloma (MM). In this study, we explored the role of miR-21 in human MM cells and searched for miR-21 targets. By knocking down the expression of endogenous miR-21 in U266 myeloma cells, we observed reduced growth, an arrested cell cycle, and increased apoptosis. To further understand its molecular mechanism in the pathogenesis of MM, we employed a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic strategy to systematically identify potential targets of miR-21. In total, we found that the expression of 178 proteins was up-regulated significantly by miR-21 inhibition, implying that they could be potential targets of miR-21. Among these, the protein inhibitor of activated STAT3 (PIAS3) was confirmed as a direct miR-21 target by Western blotting and reporter gene assays. We further demonstrated that miR-21 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3 and that down-regulation of PIAS3 contributes to the oncogenic function of miR-21. This elucidation of the role of PIAS3 in the miR-21-STAT3 positive regulatory loop not only may shed light on the molecular basis of the biological effects of miR-21 observed in MM cells but also has direct implications for the development of novel anti-MM therapeutic strategies.
Journal of Proteome Research 02/2012; 11(4):2078-90. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells.
PLoS ONE 01/2012; 7(1):e29554. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microcystin-RR (MC-RR) is a commonly encountered cyanotoxin and receives increasing attention due to the risk of its bioaccumulation in aquatic animals like fish. This study investigated the protein profiles of zebrafish (Danio rerio) testes after intraperitoneal injection (i.p.) with 0.5 LD(50) (2000 μg/kg). MC-RR caused a noticeable damage to testicular ultrastructure, showing widened intercellular junction, distention of mitochondria. The testes showed a rapid response of its defense systems to the oxidative stress caused by MC-RR. This is the first to use a proteomic approach to obtain an overview of the effects of MC-RR on the testes of zebrafish. The proteomic results revealed that toxin exposure remarkably altered the abundance of 24 proteins that were involved in cytoskeleton assembly, oxidative stress, glycolysis metabolism, calcium ion binding and other biological functions. In conclusion, MC-RR damaged the testes and was toxic to the reproductive system of male zebrafish mainly through causing oxidative stress.
[show abstract][hide abstract] ABSTRACT: Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved in the pathogenesis of various human diseases and is constitutively active in human multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry analysis allowed us to distinguish specific Stat3 interacting proteins from background proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting proteins - 14-3-3ζ, PRKCB and Hsp90 - were further confirmed by reciprocal co-immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results therefore not only uncover a number of Stat3 interacting proteins that possess a variety of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells.
Journal of proteomics 10/2011; 75(3):1055-66. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evaluation of: Rajpal R, Dowling P, Meiller J et al. A novel panel of protein biomarkers for predicting response to thalidomide-based therapy in newly diagnosed multiple myeloma patients. Proteomics 11(8), 1391-1402 (2011). Predicting response to thalidomide-based therapy remains a challenging task faced by clinicians in the treatment of multiple myeloma. The pioneering work reported by Rajpal et al. moves one step further towards solving this challenge. They developed a proteomics-based approach that combines immunodepletion, 2D-difference gel electrophoresis analysis and mass spectrometry to search for serum proteins with expressions that show significant correlations to thalidomide treatment. This integrated approach allowed them to identify a panel of protein biomarkers. By using ELISA-based validation and strict statistical analysis, the authors have achieved an overall 84.0% predictive accuracy, with associated sensitivity and specificity values of 81.8 and 86.2%, respectively. Their methods and significant findings are reviewed within this article. This panel of biomarkers may not only guide initial therapy, but can also provide direct implications for personalized medicine in multiple myeloma patients.
Expert Review of Proteomics 08/2011; 8(4):439-42. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: As cynobacterial blooms frequently occur in fresh waters throughout the world, microcystins (MCs) have caused serious damage to both wildlife and human health. MCs are known to have developmental toxicity, however, the possible molecular mechanism is largely unknown. This is the first toxicological study to integrate post-transcriptomic, proteomic and bioinformatics analysis to explore molecular mechanisms for developmental toxicity of MCs in zebrafish. After being microinjected directly into embryos, MC-RR dose-dependently decreased survival rates and increased malformation rates of embryos, causing various embryo abnormalities including loss of vascular integrity and hemorrhage. Expressions of 31 microRNAs (miRNAs) and 78 proteins were significantly affected at 72 hours post-fertilisation (hpf). Expressions of miR-430 and miR-125 families were also significantly changed. The altered expressions of miR-31 and miR-126 were likely responsible for the loss of vascular integrity. MC-RR significantly reduced the expressions of a number of proteins involved in energy metabolism, cell division, protein synthesis, cytoskeleton maintenance, response to stress and DNA replication. Bioinformatics analysis shows that several aberrantly expressed miRNAs and proteins (involved in various molecular pathways) were predicted to be potential MC-responsive miRNA-target pairs, and that their aberrant expressions should be the possible molecular mechanisms for the various developmental defects caused by MC-RR.
PLoS ONE 01/2011; 6(7):e22676. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expression of glutathione S-transferase (GST) is a crucial factor in determining the sensitivity of cells and organs in response to a variety of toxicants. In this study, we cloned the core nucleotide of alpha, kappa, mu, mGST, pi, rho, and theta-like GST genes from bighead carp (Aristichthys nobilis). Their derived amino acid sequences were clustered with other vertebrate GSTs in a phylogenetic tree, and the bighead carp GST sequences have the highest similarity with those from common carp and zebrafish. We quantified the constitutive mRNA transcription of GST isoforms in eight different tissues (liver, kidney, spleen, intestine, muscle, heart, brain, and gill). The information obtained from the present study could be distilled into a few generalized principles: multiple GST isoenzymes were ubiquitously expressed in all tissues; majority of GSTs had high constitutive expression in intestine, liver, and kidney. These findings are in agreement with the roles of these tissues in xenobiotic metabolism. At the same time, some unique findings were detected in the current experiment: (1) higher expression of most GSTs was observed in spleen; (2) the expression of GST pi was highest in almost all the studied tissues except muscle; the other two isoforms, GST alpha and rho, were also highly expressed in liver, kidney, intestine, spleen, heart, and brain of bighead carp. All these results strongly imply an important role of these GST isoforms in detoxification of ingested xenobiotics.
Journal of Biochemical and Molecular Toxicology 02/2010; 24(4):250-9. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study explored the mechanisms of toxicity of microcystins by measuring the transcription levels of nine cytoskeletal genes (actin, tubulin, vimentin, ezrin, radixin, moesin, MAP1b, tau, stathmin) in the liver, kidney and spleen of male Wistar rats treated with microcystins at a dose of 80 microg MC-LReq kg(-1) bw. Microcystins disrupted the transcriptional homeostasis of cytoskeletal genes in these organs. Changes in the transcription of four genes (beta-actin, ezrin, radixin and tau) in liver, one gene (stathmin) in kidney, and one gene (radixin) in spleen were significantly correlated with the tissue concentration of microcystins. However, the influences on the transcription of most genes we studied were greater in the liver than in the kidney or spleen. The effects of microcystins on the transcription of cytoskeletal genes may explain some of the morphological and pathological changes observed in these organs and provide new information on the hepatotoxicity of these compounds. Additionally, transcriptional changes in tumor-associated cytoskeletal genes (ezrin, moesin and stathmin) that were observed in the present study provide a possible clue to the tumor-promoting potential of microcystins and their influences on the transcription of MAP1b and tau imply possible neurological toxicity of microcystins in vertebrates.
[show abstract][hide abstract] ABSTRACT: Microcystins (MCs) induce the production of reactive oxygen species (ROS) in various tissues in mammals, whereas the endogenous antioxidant enzymes are responsible to scavenge the ROS. ROS can modulate the antioxidant enzyme activities by regulating the mRNA levels. The present study was undertaken to find out the relationship between the transcriptional alterations of antioxidant enzymes and MCs stimulation in rats. The time-dependent changes of relative transcription abundance of catalase (CAT), Mn-superoxide dismutase (Mn-SOD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and gamma-glutamylcysteine synthetase (γ-GCS) were investigated in three organs (liver, kidney, and testis) of male Wistar rats injected intravenously (i.v.) with 80 μg MC-LR(equivalent)/kg body weight using the quantitative real-time PCR (qPCR) method. We found that MCs could affect the transcriptional activities of these antioxidant enzymes in liver, kidney, and testis of MCs-treated rats and we speculated the possible causation of the transcriptional change. The altered transcription of antioxidant enzymes may play an important role in counteracting the potential deleterious effects of elevated oxidative stress induced by MCs, and this will provide us new insights into the possible role of antioxidant enzymes in the toxicological mechanisms of MCs at molecular level.
Journal of Biochemical and Molecular Toxicology 01/2010; 24(6):361-7. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: The glutathione S-transferases (GST) play important roles in the detoxification of microcystins (MCs). For better understanding of the responses of GST isforms to MCs exposure, informations about the effects of MCs on GSTs are necessary. In this experiment, we cloned the full length cDNA of 14 GST isoforms (GST alpha, kappa, mu, omega, pi, theta, zeta, and microsomal GST) from Wistar rat. The mRNA abundance of each rat GST isoform in the liver, kidney, and testis was analyzed by real time quantitative PCR. Multiple GST isoforms were constitutively expressed in all examined organs, but some isoforms were expressed at higher level in one organ than in others. The relative changes of the mRNA abundance in the liver, kidney, and testis of Wiatar rat i.v. injected with crude MCs extract at dose of 1LD(50) were also analyzed. Generally, the expression of most GSTs in the liver and testis was suppressed while that in kidney was induced after being injected with MCs. It is suggested that the transcription of GST isoforms varied in different ways within an organ and between organs of Wistar rat exposed to MCs.
[show abstract][hide abstract] ABSTRACT: It has been reported that microcystins (MCs) could accumulate in the gonads of mammals and MCs exposure exerts obvious toxic effects on male reproductive system of mammals. We have comfirmedthat MCs could accumulate and induce apoptosis in rat testis. The p53, Bax, and Bcl-2 protein play important roles in mitochondria-dependent apoptotic pathway, and this study aimed to investigate whether the p53, Bax, and Bcl-2 pathway is involved in microcystins-induced apoptosis in rat testis and discussed the possible mechanisms. Our results show that MCs led to persistent increase of transcriptional and protein level of P53 and Bax expression but led to decrease of Bcl-2 expression, resulting in an increased ratio of Bax to Bcl-2, which might contribute to apoptotic cell death of rat testis following MCs treatment. The increased ratio of expression of Bax to that of Bcl-2 induced by MCs suggests their important role in MCs-induced apoptosis in rat testis tissue.
[show abstract][hide abstract] ABSTRACT: Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from 1 h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly.
[show abstract][hide abstract] ABSTRACT: In the present research, the changes of ultrastructures and biochemical index in rabbit testis were examined after i.p. injection with 12.5 microg/kg microcystin (MC) extracts. Ultrastructural observation showed widened intercellular junction, distention of mitochondria, endoplasmic reticulum, and Golgi apparatus. All these changes appeared at 1, 3, and 12 h, but recovered finally. In biochemical analyses, the levels of lipid peroxidation (MDA) and H(2)O(2) increased significantly at 1 h, indicating MC-caused oxidative stress. Finally, H(2)O(2) decreased to the normal levels, while MDA remained at high levels. The antioxidative enzymes (CAT, SOD, GPx, GST) and antioxidants (GSH) also increased rapidly at 1 h, demonstrating a quick response of the defense systems to the oxidative stress. Finally, the activity of CAT, SOD, and GPX recovered to the normal level, while the activity of GST and the concentration of GSH remained at a high level. This suggests that the importance of MCs detoxification by GST via GSH, and the testis of rabbit contained abundant GSH. The final recovery of ultrastructure and some biochemical indexes indicates that the defense systems finally succeeded in protecting the testis against oxidative damage. In conclusion, these results indicate that the MCs are toxic to the male rabbit reproductive system and the mechanism underlying this toxicity might to be the oxidative stress caused by MCs. Although the negative effects of MCs can be overcome by the antioxidant system of testis in this study, the potential reproductive risks of MCs should not be neglected because of their wide occurrence.